A complete bioconversion cascade for dehalogenation and denitration by bacterial flavin–dependent enzymes

Abstract

Edited by F. Peter GuengerichHalogenated phenol and nitrophenols are toxic compounds that are widely accumulated in the environment. Enzymes in the had operon from the bacterium Ralstonia pickettii DTP0602 have the potential for application as biocatalysts in the degradation of many of these toxic chemicals. HadA monooxygenase previously was identified as a two-component reduced FAD (FADH ؊ )-utilizing monooxygenase with dual activities of dehalogenation and denitration. However, the partner enzymes of HadA, that is, the flavin reductase and quinone reductase that provide the FADH ؊ for HadA and reduce quinone to hydroquinone, remain to be identified. In this report, we overexpressed and purified the flavin reductases, HadB and HadX, to investigate their functional and catalytic properties. Our results indicated that HadB is an FMN-dependent quinone reductase that converts the quinone products from HadA to hydroquinone compounds that are more stable and can be assimilated by downstream enzymes in the pathway. Transient kinetics indicated that HadB prefers NADH and menadione as the electron donor and acceptor, respectively. We found that HadX is an FAD-bound flavin reductase, which can generate FADH ؊ for HadA to catalyze dehalogenation or denitration reactions. Thermodynamic and transient kinetic experiments revealed that HadX prefers to bind FAD over FADH؊ and that HadX can transfer FADH ؊ from HadX to HadA via free diffusion. Moreover, HadX rapidly catalyzed NADH-mediated reduction of flavin and provided the FADH ؊ for a monooxygenase of a different system. Combination of all three flavin-dependent enzymes, i.e. HadA/HadB/HadX, reconstituted an effective dehalogenation and denitration cascade, which may be useful for future bioremediation applications. 2 The abbreviations used are: HPs, halogenated phenol; HadA, two-component FADH Ϫ utilizing monooxygenase; HadB, a quinone reductase encoded in the had operon; HadX, a flavin reductase encoded in the had operon; NPs, nitrophenols; FADH Ϫ , reduced flavin adenine dinucleotide; FMNH Ϫ , reduced flavin mononucleotide; 4-NP, 4-nitrophenol; 4-CP, 4-chlorophenol; HQ, hydroquinone; BQ, benzoquinone; C 1 , the flavin reductase component of p-hydroxyphenylacetate 3-hydroxylase from A. buamannii; C 2 , the oxygenase component of p-hydroxyphenylacetate 3-hydroxylase from A.

Microbial degradation of halogenated aromatics: molecular mechanisms and enzymatic reactions

“…D. Oxidative catabolism of 2,4,5‐trichlorophenol from Ralstonia pickettii DTP 0602 involves Had enzymes. HadA and HadX are the two‐component flavin‐dependent monooxygenase and reductase, respectively, while HadB is a quinone reductase (Pimviriyakul et al ., ; Pimviriyakul and Chaiyen, ). E. Chlorobenzene biodegradation pathway from Pseudomonas putida strain GJ 31 occurs via a dioxygenation reaction by chlorobenzene dioxygenase (CbzA) and chlorobenzene dihydrodiol dehydrogenase (CbzB) to generate 3‐chlorocatechol as a product.…”

“…For a single‐component enzyme like PcpB from S. chlorophenolicum , the substrate is pentachlorophenol and the flavin is reduced via the blue pathway (Hlouchova et al ., ; Rudolph et al ., ). For two‐component enzymes, the HadA‐HadX system from R. pickettii , the substrate is 4‐chlorophenol and the flavin is reduced via the red pathway where HadA is a flavin‐dependent monooxygenase and HadX is a flavin reductase (Pimviriyakul et al ., ; Pimviriyakul and Chaiyen, ). B represents a catalytic base for proton abstraction to facilitate electrophilic aromatic substitution.…”

“…Two‐component monooxygenase‐reductase systems found in various aerobic microbes include the HadA‐HadX system from Ralstonia pickettii DTP006 (blue reaction in Fig. D) (Hatta et al ., ; Pimviriyakul and Chaiyen, ), the TcpA‐TcpX system from Ralstonia eutropha JMP134 (blue reaction in Fig. A) (Matus et al ., ; Xun and Webster, ; Belchik and Xun, ), the TftD‐TftC system from Burkholderia cepacia AC1100 (Gisi and Xun, ; Webb et al ., ), the CphC‐I‐CphB system from Arthrobacter chlorophenolicus A6 (Cho et al ., ; Kang et al ., ), the DcmB1‐DcmB2 system from Rhodococcus sp .…”

“…These enzymatic systems catalyse the hydroxylation and halide elimination of halogenated phenol derivatives at the para‐ or ortho‐ positions to yield either hydroquinone or catechol derivatives as products. It should be mentioned that in addition to dehalogenation, some of these systems can also catalyse elimination reactions of other substituents including denitration of nitrophenol by HadA‐HadX (Pimviriyakul and Chaiyen, ; Pimviriyakul et al ., ) and by CphC‐I‐CphB (Kang et al ., ).…”

Creating Flavin Reductase Variants with Thermostable and Solvent‐Tolerant Properties by Rational‐Design Engineering

“…In the case of the two‐component flavin‐dependent monooxygenases, in general, the reduced flavin generated by a flavin reductase must be transferred to a corresponding monooxygenase to complete the hydroxylation reaction. Therefore, key biological processes such as catabolism, detoxification, biosynthesis, and light emission often involve coupled reductase‐ and monooxygenase‐catalyzed reactions …”